Neurodegenerative disorders and methods of treatment and diagnosis thereof

ABSTRACT

This invention relates to compositions and methods for the therapy and diagnosis of neurodegenerative or neuromuscular disorders. More particularly, this invention relates to use of anaplerotic agents for treating, preventing, or delaying the onset of a neurodegenerative or neuromuscular disorder.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 61/904,365, filed Nov. 14, 2013, which is hereby incorporated byreference in its entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entireties: A computerreadable format copy of the Sequence Listing (filename:ULPI_018_01WO_SeqList_ST25.txt, date recorded Nov. 12, 2014, file size 4kilobytes).

TECHNICAL FIELD

THIS INVENTION relates generally to compositions and methods for thetherapy and diagnosis of neurodegenerative and/or neuromusculardisorders. More particularly, this invention relates to use ofanaplerotic agents for treating, preventing and/or delaying the onset ofa neurodegenerative and/or neuromuscular disorder.

BACKGROUND

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative diseasecharacterized by the progressive loss of motor neurons and concomitantmuscle wasting. This results in increased paralysis and death within 2-5years after diagnosis (Wijesekera and Leigh, 2009). The cause of deathof motor neurons is unclear, but abnormal mitochondria, ubiquinatedinclusions and neurofilament aggregates are thought to contribute.

Mitochondria are responsible for beta-oxidation of fatty acids andoxidative phosphorylation by the tricarboxylic acid (TCA) cycle and theelectron transport chain, producing most of the adenosine triphosphate(ATP), the primary cellular energy source that is necessary for cellfunction and survival. Thus, impaired mitochondrial function in motorneurons and muscles would result in defective energy metabolism and areduced capacity to produce ATP.

Triheptanoin, the triglyceride of heptanoate (C7 fatty acid), is a novelmetabolic therapeutic that is being used in the USA to treat patientswith rare genetic metabolic disorders of fatty acid oxidation(Brunengraber and Roe, 2006, Roe and Mochel, 2006). Triheptanoinprovides the body with heptanoate, which as a medium chain fatty aciddiffuses into the mitochondria to be metabolized to propionyl-CoA bybeta-oxidation. Alternatively heptanoate is metabolized in the liver tothe C5 ketones, β-hydropentanoate and β-ketopentanoate, both of whichare taken up by cells by monocarboxylate transporters. Carboxylation ofpropionyl-CoA produces methyl-malonyl-CoA, which can be metabolized tosuccinyl-CoA, resulting in anaplerosis—the refilling of deficient C4(containing four carbons) intermediates of the TCA cycle (FIG. 1).Anaplerotic enzymes include pyruvate carboxylase (Pcx) producingoxaloacetate in neurons and muscle, and most importantly in muscleglutamic pyruvic transaminases 1 and 2 (Gpt1 and 2), which catalyze thereaction pyruvate+glutamate<=>α-ketoglutarate+alanine and propionyl-CoAcarboxylase subunit A (Pcca) and B (Pccb) and methylmalonyl-CoA mutase(Mut, FIG. 1).

Superoxide dismutase 1 (SOD1) protein mutations have been found inapproximately 20% of patients with familial ALS and in a subset ofpatients with sporadic ALS. While usually found in the cytosol, mutantSOD1 accumulates within mitochondria and appears to contribute to manyof the mitochondrial perturbations found in ALS (Turner and Talbot,2008, Vucic and Kieman, 2009, Shi et al., 2010, Milani et al., 2011,Cozzolino and Carri, 2012). Mice overexpressing mutations in SOD1 arecurrently one of the best animal models for ALS.

SUMMARY

The present invention is based in part on the discovery that the TCAcycle plays a role in the pathogenesis of the neurodegenerative disorderALS, wherein anaplerotic agents can influence the onset and/orprogression of disease.

One form of the present invention is broadly directed to methods thatutilize anaplerotic agents for treating or preventing and/or delayingthe onset of neurodegenerative and/or neuromuscular disorders.

In a first aspect, the invention provides a method of treating an animalwith a neurodegenerative and/or neuromuscular disease, disorder orcondition, wherein said method includes the step of administering atherapeutically effective amount of one or more anaplerotic agents tosaid animal, to thereby treat the neurodegenarative and/or neuromusculardisease, disorder or condition in said animal.

In a second aspect, the invention provides a method of preventing and/ordelaying the onset of a neurodegenerative and/or neuromuscular disease,disorder or condition, wherein said method includes the step ofadministering a therapeutically effective amount of one or moreanaplerotic agents to said animal.

In a third aspect, the present invention provides triheptanoin for usein the preventative, prophylactic and/or therapeutic treatment of aneurodegenerative and/or neuromuscular disease, disorder or condition inan animal in need thereof.

In a fourth aspect, the present invention provides a kit for use in thepreventative, prophylactic and/or therapeutic treatment of an animalwith a neurodegenerative and/or neuromuscular disease, disorder orcondition, said kit comprising: (i) a therapeutically effective amountof one or more anaplerotic agents; and (ii) instructions for use.

In one embodiment, the neurodegenerative and/or neuromuscular disease,disorder or condition of any one of the aforementioned aspects is amotor neuron disease (MND).

In another embodiment, the MND is amyotrophic lateral sclerosis (ALS),primary lateral sclerosis (PLS), progressive muscular atrophy (PMA),progressive bulbar palsy (PBP), pseudobulbar palsy or spinal muscularatrophy (SMA).

In yet another embodiment, the MND is amyotrophic lateral sclerosis.

In one embodiment, the anaplerotic agents of the aforementioned aspectsare glutamate, glutamine, pyruvate and/or one or more precursors ofpropionyl-CoA.

In another embodiment, the one or more precursors of propionyl-CoA areselected from the group consisting of an uneven chain fatty acid, atriglyceride, a C5 ketone body, a phospholipid, a branched amino acidand combinations thereof.

In other embodiments, the one or more precursors of propionyl-CoA is atriglyceride or phospholipid of an uneven chain fatty acid.

In yet another embodiment, the C5 ketone body is selected from(3-hydroxypentanoate and β-ketopentanoate.

In one embodiment, the precursor of propionyl-CoA ispropionyl-carnitine.

In another embodiment, the branched amino acid is selected from thegroup consisting of valine, isoleucine and combinations thereof.

In yet another embodiment, the one or more precursors of propionyl-CoAis one or more compounds of Formula I.

wherein

R₁, R₂ and R₃ are independently selected from alkyl, alkenyl or alkynyl.

In one embodiment, R₁, R₂ and R₃ are independently selected from C₁ toC₂₀ alkyl, alkenyl or alkynyl.

In other embodiment, R₁, R₂ and R₃ are independently selected from C₃ toC₁₅ alkyl, alkenyl or alkynyl.

In yet another embodiment, R₁, R₂ and R₃ are independently selected fromC₅ to C₁₂ alkyl, alkenyl or alkynyl.

In still another embodiment, R₁, R₂ and R₃ are independently selectedfrom C₆ to C₉ alkyl, alkenyl or alkynyl.

In one embodiment, R₁, R₂ and R₃ are independently selected from methyl,ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl,undecyl, dodecyl, tridecyl, tetradecyl and pentadecyl, inclusive of allisomers.

In another embodiment, the compound of Formula I is triheptanoin.

In other embodiments, the compound of Formula I is trinonanoin.

In yet other embodiments, the compound of Formula I is tripentanoin.

In other embodiments, the one or more anaplerotic agents are provided tothe animal in an amount comprising at least about 5% of the dietarycaloric intake for the animal.

In one embodiment, the one or more anaplerotic agents are provided tothe animal in an amount comprising at least about 20% of the dietarycaloric intake for the animal.

In another embodiment, a anaplerotic agent is provided to the animal inan amount comprising at least about 30% of the dietary caloric intakefor the animal.

In yet another embodiment, the one or more anaplerotic agents areprovided to the animal in an amount comprising at least about 35% of thedietary caloric intake for the animal.

In one embodiment, the animal is a mammal.

In another embodiment, the mammal is a human.

Another form of the present invention is broadly directed to methodsthat detect alterations, changes or differences in gene expressionassociated with neurodegenerative and/or neuromuscular disorders.

In a fifth aspect, the invention provides a method of determiningwhether an animal has, or is predisposed to, a neurodegenerative and/orneuromuscular disease, disorder or condition, wherein the methodincludes the step of measuring (i) the expression level of one or morenucleic acids that respectively encode enzymes associated with energymetabolism, (ii) the expression level and/or activity of said enzyme(s)and/or (iii) the level of one or more metabolites associated with energymetabolism.

In one embodiment, a decrease or reduction in the expression level ofone or more nucleic acids that respectively encode enzymes associatedwith energy metabolism and/or a decrease or reduction in the expressionlevel and/or activity of said enzymes and/or a decrease or reduction inthe level of one or more metabolites associated with energy metabolismindicates that said animal has, or is predisposed to, theneurodegenerative and/or neuromuscular disease, disorder or condition.

In a sixth aspect, the invention provides a method of monitoring theresponse of an animal to treatment or prevention of a neurodegenerativeand/or neuromuscular disease, disorder or condition by administration ofone or more anaplerotic agents described herein, wherein the methodincludes the step of measuring (i) the expression level of one or morenucleic acids that respectively encode enzymes associated with energymetabolism (ii) the expression level and/or activity of said enzyme(s)and/or (iii) the level of one or more metabolites associated with energymetabolism.

In one embodiment, an increase in the expression level of one or morenucleic acids that respectively encode enzymes associated with energymetabolism and/or an increase in the expression level and/or activity ofsaid enzymes and/or an increase in the level of one or more metabolitesassociated with energy metabolism indicates that said animal isresponding to the administration of the one or more anaplerotic agents.

According the to the fifth and sixth aspects, the enzymes or metabolitesmay be selected from the group consisting of glycolytic enzymes ormetabolites, TCA cycle enzymes or metabolites and anaplerotic enzymes ormetabolites.

In some embodiments, the glycolytic enzyme is pyruvate dehydrogenasealpha 1, phosphoglucose isomerase (PGI), or phosphofructokinase (PFK).

In another embodiment, the TCA cycle enzymes are selected from the groupconsisting of oxoglutarate dehydrogenase and succinate dehydrogenasecomplex subunit A.

In another embodiment, the anaplerotic enzymes are selected from thegroup consisting of glutamate-pyruvate transaminase 1,glutamate-pyruvate transaminase 2, propionyl-CoA carboxylase subunit Aand B and methylmalonyl-CoA mutase.

Throughout this specification, unless the context requires otherwise,the words “comprise”, “comprises” and “comprising” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts simplified TCA cycle and anaplerosis in CNS and muscle.The numbers enclosed in boxes (“1”, “2”, and 3”) indicate anapleroticpathways that can refill the C4 intermediates of the cycle: 1. pyruvatecarboxylase (mostly in the CNS); 2. propionyl-CoA carboxylase; and 3.glutamic pyruvic transaminases (very important in muscle). C5 ketones,isoleucine (Ile), valine (Val), and heptanoate, are metabolised topropionyl-CoA and can therefore be anaplerotic via the propionyl-CoAcarboxylation pathway. OAA—oxaloacetate, 2-OG—2-oxoglutarate.

FIG. 2 shows alterations in metabolism in m. gastrocneminus ofhSOD1^(G93A) mice at mid and end stage of disease. Panel A depicts themetabolite levels of lactate or glucose in wild-type (WT) orhSOD1^(G93A) (SOD) mice. Lactate levels are decreased at mid stage(110-130 days) in the SOD mice as compared to the WT mice, indicatingreduced glycolysis, while there is an increase in glucose at end stage(150-175 days) in the SOD mice as compared to the WT mice, indicatingdecreased glucose metabolism. Panel B depicts the enzyme activity levelsof several enzymes in wild-type (WT) or hSOD1^(G93A) (SOD) mice at middisease (110-130 days). The data showreduction in maximal enzymeactivities in the m. gastrocneminus in the SOD mice as compared to theWT mice. PGI-phosphoglucose isomerase—28.5% decrease,PFK—phosphofructokinase—53% decrease, OGDH—oxoglutaratedehydrogenase—25% decrease. * p<0.05 t-tests. N=5-8 mice/group.

FIGS. 3A-3E show that triheptanoin treatment delays the loss of hindlimb grip strength in hSOD1^(G93A) mice. FIGS. 3A and 3B depict the hindlimb grip strength of wild-type (FIG. 3A) or hSOD1^(G93A) (FIG. 3B) micefed with control diet (CON) or triheptanoin (TRIH) diet. The data inFIG. 3A show that no differences in grip strength were evident betweentriheptanoin (green open triangles, n=15) and control fed wild-type mice(black filled squares, n=12). The data in FIG. 3B show that the gripstrength over time differed in triheptanoin (red crosses; n=8) vs.control diet (blue empty circles, n=5) fed high copy number transgenehSOD1^(G93A) mice (p=0.0⁴, two way ANOVA), with a significantly highergrip strength at 18 and 19.5 weeks (p<0.05 Bonferroni post-hoc test).FIG. 3C depicts the average hind limb grip strength of hSOD1^(G93A) micefed with control diet (CON) or triheptanoin (TRIH) diet. The data inFIG. 3C show that overall higher hind limb grip strength was seen in thearea under the curve over time in triheptanoin fed hSOD1^(G93A) mice(n=8) when compared to control fed hSOD1^(G93A) mice (n=5, p<0.05;t-test). FIG. 3D depicts the onset of hind limb grip strength loss (inweeks) in hSOD1^(G93A) mice fed with control diet (CON) or triheptanoin(TRIH) diet. The data in FIG. 3D show that the onset of hind limb gripstrength loss was delayed by 2.8 weeks in triheptanoin-fed hSOD1^(G93A)mice (n=8) when compared to control fed hSOD1^(G93A) mice (n=5, p=0.002;t-test). FIG. 3E depicts the ages of hSOD1^(G93A) mice at the onset ofhind limb grip strength loss (in weeks) plotted against hSOD1^(G93A)transgene copy numbers in those mice. The data in FIG. 3E show that thelinear regressions between the age of strength loss beginning againsttransgene copy numbers are significantly different between the groupsfed control diet (R²=0.89) vs. triheptanoin (R²-0.91). Specifically, thex and y intercepts are different between the two regression lines(p<0.001), but not the slopes (p=0.90).

FIG. 3F depicts the onset of balance loss on the rota-rod (in weeks) inhSOD1^(G93A) mice fed with control diet (CON) or triheptanoin (TRIH)diet. The data in FIG. 3F shows that the onset of balance loss intriheptanoin fed versus control fed SOD1^(G93A) mice was significantlydelayed by 13 days (p=0.0016).

FIG. 3G depicts the body weights of wild-type (WT) or hSOD1^(G93A) (SOD)mice fed with control diet (CON) or triheptanoin (TRIH) diet plottedagainst the ages of the mice. The data in FIG. 3G show that the bodyweights over time were significantly different between triheptanoin vs.control diet fed wild type mice and wild type mice on control diet vs.hSOD1^(G93A) mice on either diet. FIG. 3H depicts the weight loss onsetof hSOD1^(G93A) mice fed with control diet (CON) or triheptanoin (TRIH)diet. The data in FIG. 3H show that the onset of body weight loss intriheptanoin fed (n=8) compared to control fed SOD1^(G93A) mice (n=5)was delayed by 11 days (p=0.0076, t-test). * p<0.05, ** p<0.01

FIG. 4 depicts the result of motor neuron counting assay of 10 week oldwild-type (WT) or hSOD1^(G93A) (SOD) mice fed with control diet (CON) ortriheptanoin (TRIH) diet. The data in FIG. 4 show that triheptanoinalleviated motor neuron death in hSOD1^(G93A) mice fed triheptanoin(TRIH). The statistically significant loss of 37% of motor neuronsbetween the L2 and L5 region in mice fed control diet (CON, n=3) isstatistically insignificant in triheptanoin fed mice, showing 21% loss(n=4). Stereologic counts analysed by One Way ANOVAs followed by Tukey'smultiple comparisons test.

FIG. 5 depicts the result of a quantitative real time PCR analysis ofGapdh, Pdh alpha, Ogdh and Sdh subunit A mRNA of the gastrocneminusmuscle of 10 week old wild type and hSOD1^(G93A) mice fed control diet(CON) or triheptanoin (TRIH) relative to house keeping genes. Levels oftranscripts of Pdhalpha1, OGDH and SDH subunit A were reduced inhSOD1^(G93A) mice fed control diet (CON). These reductions in Pdhalpha1,and SDHalpha transcript levels were attenuated by triheptanoin feeding.One Way ANOVAs for each gene and time point, followed by Bonferroni posttests (* p<0.05, ** p<0.01) if significant.

FIG. 6 depicts relative expression levels of genes involved inanaplerosis, specifically pyruvate carboxylase Pcx, glutamic pyruvictransferases Gpt1 and 2, the alpha (Pcca) and beta (Pccb) subunit ofpropionyl-CoA carboxylase and methyl-malonyl mutase (Mut). Expression iscompared in the gastrocneminus muscle of 10 old wild type andhSOD1^(G93A) mice fed control diet (CON) or triheptanoin (TRIH) relativeto housekeeping genes. One Way ANOVAs for each gene and time point,followed by Bonferroni post tests (* p<0.05) if significant.

FIG. 7 illustrates metabolic alterations found in ALS patients andexperimental mouse models and mechanisms of action of triheptanoin. InMND patients and mouse MND models, activities of glycolysis, citratesynthase (CS), oxoglutarate dehydrogenase (OGDH) (Russell et al., 2012),and complex I activity (Allen et al., 2014); as well as the levels(Niessen et al., 2007) of oxaloacetate (OAA) and 2-oxoglutarate (2-OG)are decreased (indicated with dotted arrows next to these enzymes ormetabolites). Also, our findings of decreased maximal enzyme activitiesof PGI, PFK and OGDH in hSOD1^(G93A) mice at mid disease (110-130 days)are indicated with solid arrows next to these enzymes. Triheptanoin ismetabolised to propionyl-CoA and later to succinyl-CoA. This shouldincrease 1) TCA cycle metabolite levels, 2) oxidation of acetyl-CoA andall fuels providing acetyl-CoA, 3) the number of electrons for electrontransport chain complexes I & II, and 4) ATP production, resulting inimproved survival of neurons and muscles.

DETAILED DESCRIPTION

The present invention is based, at least in part, on the discovery thatthe TCA cycle plays a role in the pathogenesis of the neurodegenerativedisorder ALS, wherein anaplerotic agents can influence the onset and/orprogression of disease.

In broad aspects, the invention relates to methods of not only treatinga neurodegenerative and/or neuromuscular disease, disorder or condition,but also preventing and/or delaying the onset of a neurodegenerativeand/or neuromuscular disease, disorder or condition, by administering atherapeutically effective amount of one or more anaplerotic agents tosaid animal.

In another aspect, the present invention provides triheptanoin for usein the preventative, prophylactic and/or therapeutic treatment of aneurodegenerative and/or neuromuscular disease, disorder or condition.

As used herein, “treating” (or “treat” or “treatment”) refers to atherapeutic intervention that ameliorates a sign or symptom of aneurodegenerative and/or neuromuscular disease, disorder or conditionafter it has begun to develop. The term “ameliorating,” with referenceto a neurodegenerative and/or neuromuscular disease, disorder orcondition, refers to any observable beneficial effect of the treatment.Treatment need not be absolute to be beneficial to the subject. Thebeneficial effect can be determined using any methods or standards knownto the ordinarily skilled artisan.

As used herein, “preventing” (or “prevent” or “prevention” or“preventative”) and “delaying the onset” refer to a course of action(such as administering a composition comprising a therapeuticallyeffective amount of one or more propionyl-CoA precursors) initiatedprior to the onset of a symptom, aspect, or characteristic of theneurodegenerative and/or neuromuscular disease, disorder or condition soas to prevent or delay the onset of, respectively, said symptom, aspect,or characteristic. It is to be understood that such preventing need notbe absolute to be beneficial to a subject. A “prophylactic” treatment isa treatment administered to a subject who does not exhibit signs of aneurodegenerative and/or neuromuscular disease, disorder or condition,or exhibits only early signs, for the purpose of decreasing the risk ofdeveloping a symptom, aspect, or characteristic of a neurodegenerativeand/or neuromuscular disease, disorder or condition. A “therapeutic”treatment is one administered to a subject who exhibits at least onesymptom, aspect, or characteristic of the neurodegenerative and/orneuromuscular disease, disorder or condition so as to cure, remediate orreverse, at least in part, and/or halt or delay the progression of saidsymptom, aspect, or characteristic.

In the context of the present invention, by “a neurodegenerativedisease, disorder or condition” is meant any disease, disorder and/orcondition that comprises a progressive decline and/or deterioration inthe structure, function, signalling and/or population of the neurons orneural tissue in an animal. As used herein, “a neuromuscular disease,disorder or condition” refers to any disease, disorder and/or conditionthat comprises a progressive decline and/or deterioration in thestructure, function, signalling and/or population of the neurons orneural tissue that innervate and/or communicate, whether directly orindirectly, with the muscles of an animal.

The aetiology of a neurodegenerative and/or neuromuscular disease,disorder or condition may involve, but is not limited to, geneticmutations, protein misfolding and/or aggregation, autoimmune disorders,mitochondrial dysfunction, defective axonal transport, aberrantapoptosis and/or autophagy and elevated oxidative stress and/or reactiveoxygen species (ROS) production.

Without limitation, neurodegenerative disorders include Parkinson'sdisease and related disorders, Huntington's disease, Alzheimer's diseaseand other forms of dementia, Spinocerebellar ataxia, Friedreich ataxia,Tay-Sachs disease, Lewy body disease, Prion diseases (e.g.Creutzfeldt-Jakob disease), Multiple sclerosis (MS), Pick disease,Shy-Drager syndrome, pontocerebellar hypoplasia, neuronal ceroidlipofuscinoses, Gaucher disease, neurodegeneration with brain ironaccumulation, spastic ataxia/paraplegia, supranuclear palsy,mesolimbocortical dementia, thalamic degeneration,cortical-striatal-spinal degeneration, cortical-basal ganglionicdegeneration, cerebrocerebellar degeneration, Leigh syndrome, post-poliosyndrome, hereditary muscular atrophy, encephalitis, neuritis,hydrocephalus and the motor neuron diseases.

Further, the skilled artisan would understand that neuromusculardisorders may include Parkinson's disease and related disorders,Huntington's disease, Spinocerebellar ataxia, Friedreich ataxia, TaySachs disease, Lewy body disease, peripheral neuropathy, myastheniagravis, MS, Leigh syndrome, post-polio syndrome, hereditary muscularatrophy, spastic ataxia/paraplegia and the motor neuron diseases,without limitation thereto.

In the context of the present invention, the animal with aneurodegenerative and/or neuromuscular disease, disorder or conditionsubject to treatment, preventative and/or therapeutic, by the claimedmethod has been determined to either have an existing neurodegenerativeand/or neuromuscular disease, disorder or condition or be predisposed tosuch a disease, disorder or condition.

In one embodiment, the neurodegenerative and/or neuromuscular disease,disorder or condition of this broad aspect is a motor neuron disease.

Broadly, motor neuron diseases are a form of neurodegenerative and/orneuromuscular disorder that typically involve the motor neurons of anaffected animal. As would be readily understood by a skilled artisan,motor neurons are nerve cells that control the voluntary muscles of thetrunk, limbs and phalanges, as well as those muscles that influencespeech, swallowing and respiration. Accordingly, the clinical symptomsof an MND may include muscle weakness and/or wasting, muscle cramps,dysphagia, slurred speech, muscle tremors/fasciculations, reducedcognition, dyspnoea, respiratory failure, fatigue and weight losswithout limitation thereto. Motor neuron diseases include, but are notlimited to, Amyotrophic lateral sclerosis (ALS; also known as LouGehrig's disease), Primary lateral sclerosis (PLS), Progressive muscularatrophy (PMA), Progressive bulbar palsy (PBP), Pseudobulbar palsy andSpinal muscular atrophy (SMA).

In light of the foregoing, the motor neuron disease may be ALS, PLS,PMA, PBP, pseudobulbar palsy or SMA.

In one embodiment, the motor neuron disease is ALS.

By “anaplerotic agent” is meant a substance that when incorporated intothe TCA cycle either replenishes one or more depleted C4 (containingfour carbons) intermediates of the TCA cycle or maintains or increasesthe level of one or more intermediates of the TCA cycle, or both.

As the skilled artisan would appreciate, the levels of TCA cycleintermediates are crucial for the normal functioning and regulation ofthe TCA cycle. Most TCA cycle intermediates, however, are also involvedin other metabolic pathways in the cell, and consequently followingtheir efflux from the mitochondria their respective levels may be foundto be reduced in the TCA cycle. Such reductions in the levels of TCAcycle intermediates may subsequently inhibit optimal functioning of thecycle. The entry of anaplerotic agents into the TCA cycle may overcomethis efflux, and thus maintain suitable levels of TCA cycleintermediates for optimal functioning of the cycle.

In one embodiment, the one or more anaplerotic agents of the currentinvention are citrate, alpha-keto-glutarate (2-oxoglutarate), glutamate,glutamine, succinate, fumarate, malate, pyruvate and/or one or moreprecursors of propionyl-CoA.

Any relevant salts (such as calcium, sodium, magnesium or potassiumsalts), prodrugs, analogues, derivatives, substituted and/or branchedforms, precursors and derivatives of these anaplerotic agents are alsocontemplated within the scope of the invention. For example, salts ofthe present invention may include monosodium glutamate, calcium pyruvate(and calcium pyruvate monohydrate), creatine pyruvate, magnesiumpyruvate, potassium pyruvate and sodium pyruvate.

One embodiment of an anaplerotic agent is a precursor of propionyl-CoA.By “precursor of propionyl-CoA” is meant a substance from whichpropionyl-CoA can be formed by one or more metabolic reactions takingplace within a cell or tissue of an animal body.

This can include within its scope salts, prodrugs, analogues,derivatives, substituted, unsaturated, branched forms, or other unevenchain fatty acids and derivatives thereof if applicable.

Typical examples of precursors of propionyl-CoA are uneven-chain fattyacids, in particular seven-carbon fatty acids although withoutlimitation thereto, heptanoate, triglycerides inclusive of triglyceridesof an uneven chain fatty acid, a compound of Formula 1, a phospholipidcomprising one or two uneven chain fatty acid(s), C5 ketone bodies (e.g.β-ketopentanoate (3-ketovalerate), and β-hydroxypentanoate(3-hydroxyvalerate) but without limitation thereto) (Kinman 2006, Am JPhysiol Endocrinol Metab 291 (4): E860-6, Brunengraber and Roe 2006, JInherit Metabol Dis 29 (2-3): 327-31). The examples of precursors ofpropionyl-CoA described above include the compounds themselves, as wellas their salts, prodrugs, solvates and derivatives if applicable.

In some embodiments, the at least one precursor of propionyl-CoA isselected from the group consisting of an uneven chain fatty acid, atriglyceride, a phospholipid and combinations thereof.

The at least one precursor of propionyl-CoA may be an uneven-chain fattyacid, such as a seven-carbon fatty acid. In other embodiments, the atleast one precursor of propionyl-CoA is a triglyceride, such as atriglyceride of an uneven chain fatty acid. In further embodiments, theat least one precursor of propionyl-CoA is a phospholipid comprising oneor two uneven chain fatty acid(s). In some embodiments, the at least oneprecursor of propionyl-CoA is a C5 ketone bodies.

Examples of prodrugs include esters, oligomers of hydroxyalkanoate suchas oligo(3-hydroxyvalerate) (Seebach 1999, Int J Biol Macromol 25 (1-3):217-36) and other pharmaceutically acceptable derivatives, which, uponadministration to a individual, are capable of providing propionyl-CoA.A solvate refers to a complex formed between a precursor ofpropionyl-CoA described above and a pharmaceutically acceptable solvent.Examples of pharmaceutically acceptable solvents include water, ethanol,isopropanol, ethyl acetate, acetic acid, and ethanolamine. As usedherein, “derivative” compounds of the present invention have beenaltered, for example by conjugation or complexing with other chemicalmoieties.

In certain embodiments, the precursor of propionyl-CoA is an unevenchain fatty acid. The invention also includes within its scope esters ofuneven chain fatty acids. It will be appreciated by a person of skill inthe art that an uneven chain fatty acid may also be referred to as anodd-carbon number fatty acid. The uneven chain fatty acid may beselected from the group consisting of propionic acid, pentanoic acid,heptanoic acid, nonanoic acid and undecanoic acid.

Substituted, unsaturated and/or branched uneven chain fatty acids, aswell as other modified uneven chain fatty acids can be used withoutdeparting from the scope of the invention.

In other embodiments, the at least one precursor of propionyl-CoA may beone or more compounds of Formula I:

wherein

R₁, R₂ and R₃ are independently selected from alkyl, alkenyl or alkynyl.

In one embodiment, R₁, R₂ and R₃ are independently selected from C₁ toC₂₀ alkyl, alkenyl or alkynyl.

In a further embodiment, R₁, R₂ and R₃ are independently selected fromC₃ to C₁₅ alkyl, alkenyl or alkynyl.

In another further embodiment, R₁, R₂ and R₃ are independently selectedfrom C₅ to C₁₂ alkyl, alkenyl or alkynyl.

In yet another further embodiment, R₁, R₂ and R₃ are independentlyselected from C₅ to C₉ alkyl, alkenyl or alkynyl.

In certain embodiments, R1, R2 and R3 are the same and are selected fromthe group consisting of C₅, C₆, C₇, C₈ and C₉ alkyl, and in particularembodiments, selected from C₅, C₇ and C₉ alkyl and yet in anotherparticular embodiment, C₇ alkyl.

In some embodiments, R₁, R₂ and R₃ are independently selected frommethyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl,decyl, undecyl, dodecyl, tridecyl, tetradecyl and pentadecyl, inclusiveof all isomers.

R₁, R₂ and R₃ may also be independently selected from hexyl, heptyl,octyl and nonyl, inclusive of all isomers.

In particular embodiments, the compound of Formula I is an odd-numberedtriglyceride. In certain embodiments, the odd-numbered triglyceride isselected from tripentanoin, triheptanoin and trinonanoin.

In one embodiment, the compound of Formula I is a C7 triglyceridewherein it may contain one, two or three C7 chains.

In one embodiment, the compound of Formula I is triheptanoin, shownbelow. This compound may be known by a number of alternative namesincluding 1,3-di(heptanoyloxy)propan-2-yl heptanoate, 1,2,3-propanetriyltriheptanoate and glycerol triheptanoate.

In other embodiments, the compound of Formula I is trinonanoin. Thiscompound may be known by a number of alternative names includingglyceroltrinonanoate and glyceryltripelargonate.

The term “alkyl” refers to optionally substituted linear and branchedhydrocarbon groups having 1 to 20 carbon atoms. Where appropriate, thealkyl group may have a specified number of carbon atoms, for example,C₁-C₆ alkyl which includes alkyl groups having 1, 2, 3, 4, 5 or 6 carbonatoms in linear or branched arrangements. Non-limiting examples of alkylgroups include methyl, ethyl, propyl, isopropyl, butyl, s- and t-butyl,pentyl, 2-methylbutyl, 3-methylbutyl, hexyl, heptyl, 2-methylpentyl,3-methylpentyl, 4-methylpentyl, 2-ethylbutyl, 3-ethylbutyl, octyl,nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl.

The term “alkylene” refers to a saturated aliphatic chain substituted ateither end, also known as an alkanediyl. Non-limiting examples mayinclude —CH₂—, —CH₂CH₂— and —CH₂CH₂CH₂—.

The term “alkenyl” refers to optionally substituted unsaturated linearor branched hydrocarbon groups, having 2 to 20 carbon atoms and havingat least one carbon-carbon double bond. Where appropriate, the alkenylgroup may have a specified number of carbon atoms, for example, C₂-C₆alkenyl which includes alkenyl groups having 2, 3, 4, 5 or 6 carbonatoms in linear or branched arrangements. Non-limiting examples ofalkenyl groups include, ethenyl, propenyl, isopropenyl, butenyl, s- andt-butenyl, pentenyl, hexenyl, hept-1,3-diene, hex-1,3-diene,non-1,3,5-triene and the like.

The term “alkynyl” refers to optionally substituted unsaturated linearor branched hydrocarbon groups, having 2 to 20 carbon atoms and havingat least one carbon-carbon triple bond. Where appropriate, the alkynylgroup may have a specified number of carbon atoms, for example, C₂-C₆alkynyl groups have 2, 3, 4, 5 or 6 carbon atoms in linear or branchedarrangements. Non-limiting examples of alkynyl groups include ethynyl,propynyl, butynyl, penrynyl, hexynyl and the like.

In other embodiments, the invention contemplates administration of aphospholipid comprising one or two uneven chain fatty acid(s). Thephospholipid may be selected from the group consisting of phosphatidicacid (phosphatidate), phosphatidylethanolamine (cephalin),phosphatidylcholine (lecithin), phosphatidylserine andphosphoinositides.

The person skilled in the art is aware of standard methods forproduction of precursors of propionyl-CoA. A person skilled in the artis able to determine suitable conditions for obtaining the compounds asdescribed herein, for example, by reference to texts relating tosynthetic methodology, non-limiting examples of which are Smith M. B.and March J., March's Advanced Organic Chemistry, Fifth Edition, JohnWiley & Sons Inc., 2001 and Larock R. C., Comprehensive OrganicTransformations, VCH Publishers Ltd., 1989. Furthermore, selectivemanipulations of functional groups may require protection of otherfunctional groups. Suitable protecting groups to prevent unwanted sidereactions are provided in Green and Wuts, Protective Groups in OrganicSynthesis, John Wiley & Sons Inc., 3rd Edition, 1999.

In some embodiments of the invention, the precursor of propionyl-CoA isa seven carbon (C7) fatty acid source or a derivative thereof. Examplesof such seven carbon fatty acid source or derivatives thereof include,but are not limited to, triheptanoin, n-heptanoic acid, n-heptanoate, atriglyceride comprising n-heptanoic acid or comprising n-heptanoic acidand a different fatty acid such as n-pentanoic acid, n-nonanoic acid, orboth. Examples of derivatives of seven carbon fatty acid source alsoinclude, but are not limited to, 4-methylhexanoate, 4-methylhexenoate,3-hydroxy-4-methylhexanoate, 5-methylhexanoate, 5-methylhexenoate and3-hydroxy-5-methylhexanoate.

For example, one embodiment of a seven carbon fatty acid source istriheptanoin, which is a triglyceride that can be made by theesterification of three n-heptanoic acid molecules and glycerol by anymeans known in the art. Triheptanoin is also commercially availablethrough Ultragenyx Pharmaceutical, although without limitation thereto.

Another example of seven-carbon fatty acid is n-heptanoic acid.n-Heptanoic acid is a saturated straight chain seven-carbon fatty acidwith the following structure:

Heptanoic acid is found in various fusel oils in appreciable amounts andcan be extracted by any means known in the art. It can also besynthesized by oxidation of heptaldehyde with potassium permanganate indilute sulfuric acid (Ruhoff, Org Syn Coll. voIII, 315 (1943)).Heptanoic acid is also commercially available through Sigma Chemical Co.(St. Louis, Mo.).

According to the present invention, any seven carbon fatty acid sourceor derivatives thereof can be used for the treatment methods provided inthe present invention. The terms heptanoic acid, heptanoate, andtriheptanoin may be used interchangeably in the present description. Itwill be understood by those skilled in the art that heptanoic acid,heptanoate, and triheptanoin are used throughout the present descriptionas an exemplary seven-carbon fatty acid source to be used in theinvention and is intended to be illustrative of the invention, but isnot to be construed to limit the scope of the invention in any way.Substituted, unsaturated, or branched heptanoate, as well as othermodified seven-carbon fatty acid source can be used without departingfrom the scope of the invention.

In some embodiments, the C7 fatty acid source is provided in aformulation that has minimum impurity, e.g., the formulation contains atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% triheptanoin.In some other embodiments, the C7 fatty acid source has an acid valuehas an acid value of 0.1 or less mg KOH/gr, a hydroxyl value of 2.8 orless mg KOH/gr.

According to the present invention, the precursors of propionyl-CoA maybe used as single agents or in association with other therapeutic agentsor treatments. In some embodiments, they are used in combination withother therapeutic agents. For example, a precursor of propionyl-CoA maybe used in combination with a carnitine supplement, biotin, vitamin B12,or combinations thereof. An example of carnitine supplement isL-carnitine. An example of vitamin B12 is Cyanocobalamin.

By “administration” or “administering” is meant the introduction of acomposition (e.g., a composition comprising one or more anapleroticagents) into a subject by a chosen route.

The term “therapeutically effective amount” describes a quantity of aspecified agent sufficient to achieve a desired effect in a subjectbeing treated with that agent. For example, this can be the amount of acomposition comprising one or more anaplerotic agents necessary toreduce, alleviate, prevent and/or delay the onset of one or a pluralityof symptoms of a neurodegenerative and/or neuromuscular disease,disorder or condition. In some embodiments, a “therapeutically effectiveamount” is sufficient to reduce or eliminate a symptom of aneurodegenerative and/or neuromuscular disease, disorder or condition.In other embodiments, a “therapeutically effective amount” is an amountsufficient to achieve a desired biological effect, for example an amountthat is effective to reverse, at least in part, the impairedmitochondrial function associated with a neurodegenerative and/orneuromuscular disease, disorder or condition.

Ideally, a therapeutically effective amount of an anaplerotic agent isan amount sufficient to induce the desired result without causing asubstantial cytotoxic effect in the subject. The effective amount of ananaplerotic agent useful for reducing, alleviating, preventing and/ordelaying the onset of one or more symptoms of a neurodegenerative and/orneuromuscular disease, disorder or condition will be dependent on thesubject being treated, the type and severity of any associated disease,disorder and/or condition, and the manner of administration of thetherapeutic composition.

The present invention includes within its scope a therapeutic amount ofone or more anaplerotic agents that are less than 100% of dietarycaloric intake and may be within a range from between about 5% and about90%, between about 15% and about 80%, between about 20% and about 60%,between about 25% and 50% and/or between about 30% and about 40%.

In particular embodiments, the one or more anaplerotic agents areprovided to the animal in an amount comprising at least about 5%, atleast about 10%, at least about 15%, at least about 20%, at least about20.5%, at least about 21%, at least about 21.5%, at least about 22%, atleast about 22.5%, at least about 23%, at least about 23.5%, at leastabout 24%, at least about 24.5% at least about 25%, at least about25.5%, at least about 26%, at least about 26.5%, at least about 27%, atleast about 27.5%, at least about 28%, at least about 28.5%, at leastabout 29%, at least about 29.5%, at least about 30%, at least about30.5%, at least about 31%, at least about 31.5%, at least about 32%, atleast about 32.5%, at least about 33%, at least about 33.5%, at leastabout 34%, at least about 34.5%, at least about 35%, at least about35.5%, at least about 36%, at least about 36.5%, at least about 37%, atleast about 37.5%, at least about 38%, at least about 38.5%, at leastabout 39%, at least about 39.5%, at least about 40%, at least about40.5%, at least about 41%, at least about 41.5%, at least about 42%, atleast about 42.5%, at least about 43%, at least about 43.5%, at leastabout 44%, at least about 44.5%, at least about 45%, at least about45.5%, at least about 46%, at least about 46.5%, at least about 47%, atleast about 47.5%, at least about 48%, at least about 48.5%, at leastabout 49%, at least about 49.5%, at least about 50%, at least about 55%,at least about 60%, about at least about 70%, at least about 80%, atleast about 90% or more of the dietary caloric intake.

In one embodiment, the one or more anaplerotic agents are provided tothe animal in an amount comprising at least about 5% of the dietarycaloric intake for the animal.

In a further embodiment, the one or more anaplerotic agents are providedto the animal in an amount comprising at least about 20% of the dietarycaloric intake for the animal.

In another further embodiment, the one or more anaplerotic agents areprovided to the animal in an amount comprising at least about 30% of thedietary caloric intake for the animal.

In yet another further embodiment, the one or more anaplerotic agentsare provided to the animal in an amount comprising at least about 35% ofthe dietary caloric intake for the animal.

It will be appreciated by a skilled addressee that “% of dietary caloricintake” may relate to % of kJoules or % of kcal.

Any safe route of administration may be employed for providing a patientwith the one or more anaplerotic agents. For example, enteral, oral,rectal, parenteral, sublingual, buccal, intra-duodenal, intra-gastric,intravenous, intra-articular, intra-muscular, intra-dermal,subcutaneous, inhalational, intraocular, intraperitoneal,intracerebroventricular, transdermal and the like may be employed. Itcan be administered via ingestion of a food substance containingtriheptanoin at a concentration effective to achieve therapeutic levels.Alternatively, it can be administered as a capsule or entrapped inliposomes, in solution or suspension, alone or in combination with othernutrients, additional sweetening and/or flavoring agents. Capsules andtablets can be coated with shellac and other enteric agents as is known.

Dosage forms include tablets, dispersions, suspensions, injections,solutions, syrups, oils troches, capsules, suppositories, aerosols,transdermal patches and the like. These dosage forms may also includeinjecting or implanting controlled releasing devices designedspecifically for this purpose or other forms of implants modified to actadditionally in this fashion. Controlled release of the therapeuticagent may be effected by coating the same, for example, with hydrophobicpolymers including acrylic resins, waxes, higher aliphatic alcohols,polylactic and polyglycolic acids and certain cellulose derivatives suchas hydroxypropylmethyl cellulose. In addition, the controlled releasemay be effected by using other polymer matrices, liposomes and/ormicrospheres.

Anaplerotic agents for enteral, intraperitoneal, oral or parenteraladministration may be presented in discrete units such as capsules,sachets or tablets each containing a pre-determined amount of thetherapeutic agent of the invention, as a powder or granules or as asolution or a suspension in an aqueous liquid, a non-aqueous liquid, anoil-in-water emulsion or a water-in-oil liquid emulsion. In certainembodiments, administration of the one or more anaplerotic agents is byway of oral administration. Such therapeutically effective amounts ofthe one or more anaplerotic agents may be prepared by any of the methodsof pharmacy but all methods include the step of bringing intoassociation one or more agents as described above with the carrier whichconstitutes one or more necessary ingredients. In general, suchcompositions may be prepared by uniformly and intimately admixing theagents of the invention with liquid carriers or finely divided solidcarriers or both, and then, if necessary, shaping the product into thedesired presentation.

The anaplerotic agents may be administered in a manner compatible withthe dosage formulation, and in such amount as ispharmaceutically-effective. The dose administered to a patient, in thecontext of the present invention, should be sufficient to effect abeneficial response in a patient over an appropriate period of time. Thequantity of agent(s) to be administered may depend on the subject to betreated inclusive of the age, sex, weight and general health conditionthereof, factors that will depend on the judgement of the practitioner.

It will also be appreciated that treatment methods may be applicable toprophylactic or therapeutic treatment of mammals, inclusive of humansand non-human mammals such as livestock (e.g. horses, cattle and sheep),companion animals (e.g. dogs and cats), laboratory animals (e.g. micerats and guinea pigs) and performance animals (e.g. racehorses,greyhounds and camels), although without limitation thereto.

In a further aspect, the invention provides a kit for the treatment ofan animal with a neurodegenerative and/or neuromuscular disease,disorder or condition comprising: (i) a therapeutically effective amountof one or more anaplerotic agents described herein; and (ii)instructions for use.

The one or more anaplerotic agents may be selected from tripentanoin,triheptanoin and trinonanoin.

In a particular embodiment, the one or more anaplerotic agents istriheptanoin.

The one or more anaplerotic agents of this aspect may further comprise apharmaceutically-acceptable carrier, diluent or excipient. Such apharmaceutically-acceptable carrier, diluent or excipient may include asolid or liquid filler, diluent or encapsulating substance that may besafely used in systemic administration. A useful reference describingpharmaceutically acceptable carriers, diluents and excipients isRemington's Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991)which is incorporated herein by reference.

It would be appreciated that certain embodiments of this aspect mayfurther comprise one or more additional therapies for aneurodegenerative and/or neuromuscular disease, disorder and/orcondition. These may include, for example, antioxidants,anti-inflammatories, anti-apoptotic compounds, cholinesteraseinhibitors, acetylcholine receptor agonists and/or antagonists,α-adrenoceptor agonists and/or antagonists, monoamine oxidaseinhibitors, catechol-O-methyl transferase inhibitors, nitric oxidedonors, analgesics, riluzole (Rilutek®), muscle relaxants and/oranticonvulsants, albeit without limitation thereto.

The one or more anaplerotic agents of this aspect may comprise a dosageform as described hereinbefore. Furthermore, a skilled artisan wouldreadily acknowledge that certain embodiments of the present aspect mayinclude devices suitable for dispensing the dosage form/s and/orcomposition/s of said kit. Such dispensing devices may include, forexample, one or more of a syringe and/or needle, blister pack,applicator, cup or suitably shaped container, inhalant device, spoon,dropper, nebulizer, transdermal patch, gauze and/or bandage, albeitwithout limitation thereto.

In another further aspect, the invention provides a method ofdetermining whether an animal has, or is predisposed to, aneurodegenerative and/or neuromuscular disease, disorder or condition,wherein the method includes the step of measuring (i) the expressionlevel of one or more nucleic acids that respectively encode enzymesassociated with energy metabolism (ii) the expression level and/oractivity of said enzymes and/or (iii) the level of one or moremetabolites associated with energy metabolism.

In yet another further aspect, the invention provides a method ofmonitoring the response of an animal to treatment or prevention of aneurodegenerative and/or neuromuscular disease, disorder or condition byadministration of one or more anaplerotic agents, wherein the methodincludes the step of measuring (i) the expression level of one or morenucleic acids that respectively encode enzymes associated with energymetabolism (ii) the expression level and/or activity of said enzymesand/or (iii) the level of one or more metabolites associated with energymetabolism.

The neurodegenerative and/or neuromuscular disease, disorder orcondition is as hereinbefore described.

By “energy metabolism” is meant the sum total of all the chemicalreactions and/or processes involved in maintaining the energy supply ofa cell and thus an organism. These reactions and/or processes arelargely catalyzed by enzymes, and typically involve the breakdown oflarger molecules, such as carbohydrates like glucose, to smallermolecules to extract energy. For the present invention, those substancesinvolved in or produced as a result of these reactions and/or processesare referred to as metabolites. Broadly, energy metabolism includes, butis not limited to, glycolysis, the TCA cycle, oxidative phosphorylation,anaplerosis and anaerobic respiration.

In some embodiments of the methods of the invention, the enzymes ormetabolites may be selected from the group consisting of glycolyticenzymes or metabolites, TCA cycle enzymes or metabolites and anapleroticenzymes or metabolites.

Non-limiting examples of glycolytic enzymes include hexokinase,phosphoglucose isomerase, phosphofructokinase, fructose-biphosphatealdolase, triose-phosphate isomerase, glyceraldehyde phosphatedehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase,enolase, pyruvate kinase, pyruvate dehydrogenase, dihydrolipoyltransacetylase and dihydrolipoyl dehydrogenase.

In specific embodiments, the glycolytic enzyme is pyruvate dehydrogenasealpha 1, phosphoglucose isomerase (PGI), or phosphofructokinase (PFK).

Non-limiting examples of glycolytic metabolites include glucose, glucose6-phosphate, fructose 6-phosphate, fructose 1,6-biphosphate,glyceraldehyde 3-phosphate, 1,3-biphosphoglycerate, 3-phosphoglycerate,2-phosphoglycerate, phosphoenolpyruvate, pyruvate and lactate.

Non-limiting examples of TCA cycle enzymes include citrate synthase,aconitase, isocitrate dehydrogenase, oxoglutarate dehydrogenase,succinyl-CoA synthctase, succinate dehydrogenase complex, fumarase,Complex I (a.k.a. NADH dehydrogenase or NADH ubiquinone oxidoreductase),and malate dehydrogenase.

The TCA cycle enzymes may be selected from the group consisting ofoxoglutarate dehydrogenase and succinate dehydrogenase complex subunitA.

Non-limiting examples of TCA cycle metabolites include acetyl CoA,citrate, cis-Aconitate, isocitrate, oxalosuccinate, oxoglutarate,succinyl CoA, succinate, fumurate, malate and oxaloacetate.

Non-limiting examples of anaplerotic enzymes include pyruvatecarboxylase, aspartate transaminase, glutamate dehydrogenase,methylmalonyl-CoA mutase, adenylosuccinate lyase, glutamate pyruvatetransaminase 1, glutamate pyruvate transaminase 2 and propionyl-CoAcarboxylase subunits A and B.

The anaplerotic enzymes may be selected from the group consisting ofglutamate-pyruvate transaminase 1, glutamate-pyruvate transaminase 2,propionyl-CoA carboxylase subunit A and B and methylmalonyl-CoA mutase.

Non-limiting examples of anaplerotic metabolites include aspartate,alanine, GABA, citrate, succinate, malate, fumarate, alpha-ketoglutarate(2-oxo-glutarate), glutamate, glutamine, proline, histidine, arginine,phenylalanine, tyrosine, methionine, isoleucine, valine, propionyl CoA,methylmalonyl CoA and adenylosuccinate.

A person skilled in the relevant art would readily acknowledge that ifthe levels of metabolites associated with the TCA cycle and/oranaplerosis, such as lactate, alanine, pyruvate, aspartate, glutamate,glutamine and aspartate, are lowered, this indicates that there may beless TCA cycle intermediates and/or less anaplerosis.

As would be well appreciated by the skilled artisan, reference herein toan increased or decreased level of expression (e.g a nucleic acid orprotein expression level, or the level of a metabolite) or activity (e.genzyme activity) includes and encompasses relative and absolute levelsof expression. By way of example, a relative level of expression oractivity may be determined relative to a reference or standard level ofexpression or activity, such as in a sample obtained from a normal ornon-suffering individual or population of individuals.

A change or alteration in the level of a metabolite or the level of aenzyme's protein expression, gene expression and/or enzymatic activityin an animal that has, or is predisposed to, a neurodegenerative and/orneuromuscular disease, disorder or condition may be determined bycomparing such levels from a biological sample obtained from said animalto either those levels of another animal known not to have, nor bepredisposed to, a neurodegenerative and/or neuromuscular disease,disorder or condition or those levels of the general population or areference population.

In one embodiment, a decrease or reduction in the expression level ofone or more nucleic acids that respectively encode enzymes associatedwith energy metabolism and/or a decrease or reduction in the expressionlevel and/or activity of said enzyme(s) and/or a decrease or reductionin the level of one or more metabolites associated with energymetabolism indicate that said animal has, or is predisposed to, theneurodegenerative and/or neuromuscular disease, disorder or condition.

In yet another embodiment an increase in the expression level of one ormore nucleic acids that respectively encode enzymes associated withenergy metabolism and/or an increase in the expression level and/oractivity of said enzyme(s) and/or an increase in the level of one ormore metabolites associated with energy metabolism indicate that saidanimal is responding to the administration of one or more anapleroticagents.

Typically, the one or more nucleic acids that respectively encodeenzymes associated with energy metabolism and/or said enzyme(s) and/orthe one or more metabolites associated with energy metabolism aredetected or measured in a biological sample. The sample may be a fluid,cell or tissue sample obtained from the animal.

In this context, a “nucleic acid” may be single- or double-stranded DNAinclusive of genomic DNA and cDNA although without limitation thereto ormay be single- or double-stranded RNA inclusive of mRNA and miRNA,although without limitation thereto. A particular example of a nucleicacid is an “amplification product” resulting from nucleic acid sequenceamplification. Non-limiting examples of primers suitable for nucleicacid amplification of nucleic acids encoding these enzymes compriserespective nucleotide sequences set forth in Table 1.

TABLE 1Gene names, GenBank accession numbers, symbols, forward and reverseprimer sequences (SEQ ID NOS: 1-20) used for the gene expression studiesof metabolic genes. Gene Symbol Sequence 5′ to 3′ Propionyl-CoA PccaF AGAATTGCAAGGGAAATTGG Carboxylase (Subunit A) (SEQ ID NO: 1)NM_144844.2 R CTAAAGCCATCCCTGGTCTC (SEQ ID NO: 2) Propionyl-CoA PccbF AGCCTACAACATGCTGGACA Carboxylase (Subunit B) (SEQ ID NO: 3)NM_025835.2 R GGTCCTCCCATTCATTCTTG (SEQ ID NO: 4) Methylmalonyl-CoA MutF CCAAACACTGACCGTTCTCA mutase (SEQ ID NO: 5) NM_008650.3R GGAATGTTTAGCTGCTTCAGG (SEQ ID NO: 6) Pyruvate carboxylase PcxF GAGCTTATCCCGAACATCCC NM_008797.3 (SEQ ID NO: 7)R TCCATACCATTCTCTTTGGCC (SEQ ID NO: 8) Pyruvate dehydrogenase Pdha1F AACTTCTATGGAGGCAACGG (SEQ E1 alpha 1 ID NO: 9) NM_008810.2R CTGACCCTGATTAGCAGCAC (SEQ ID NO: 10) Glyceraldehyde-3- GapdhF ATACGGCTACAGCAACAGGG phosphate dehydrogenase (SEQ ID NO: 11)NM_008084.2 R TCTTGCTCAGTGTCCTTGCT (SEQ ID NO: 12) Oxoglutarate OgdhF TGCAGATGTGCAATGATGAC (SEQ dehydrogenase ID NO: 13) NM_010956.4R GCAGCACATGGAAGAAGTTG (SEQ ID NO: 14) Succinate dehydrogenase SdhaF GGAACACTCCAAAAACAGACCT complex (Subunit A) (SEQ ID NO: 15) NM_023381.1R CCACCACTGGGTATTGAGTAGAA (SEQ ID NO: 16) Glutamate-pyruvate Gpt1F TGAGGTTATCCGTGCCAATA transaminase 1 (SEQ ID NO: 17) NM_182805.2R GTCCGGACTGCTCAGAAGAT (SEQ ID NO: 18) Glutamate-pyruvate Gpt2F GCGACGGTATTTCTACAATCC transaminase 2 (alanine (SEQ ID NO: 19)aminotransferase) R CGCGGAGTACAAGGGATACT NM_173866.3 (SEQ ID NO: 20)

As are well known in the art, methods of measuring gene expression mayinclude, but are not limited to, northern blotting, nucleic acidamplification techniques (e.g. qPCR, real-time PCR and competitive PCR),high throughput expression profiling (e.g. microarrays), serial analysisof gene expression (SAGE) and RNA-seq.

Suitable techniques at measuring the expression level of enzymesassociated with energy metabolism are similarly well known. Suchtechniques may include, but are not limited to, immunoassays, (e.g.western blots), radioimmunoassays, enzyme-linked immunosorbent assays(ELISAs), protein microarray techniques (e.g. reverse phase proteinmicroarray (RPPA), histological methods (e.g. immunofluorescence (IF)and immunohistochemistry (IHC)), colorimetric/fluorometric/luminescenceassays and proteomics approaches such as mass spectrometry (MS) andhigh-performance liquid chromatography (HPLC).

Suitable techniques for measuring the level of metabolites associatedwith energy metabolism are also well known in the art. Such techniquesmay include, but are not limited tocolorimetric/fluorometric/luminescence enzymatic assays,high-performance liquid chromatography (HPLC), mass spectrometry (MS)coupled with gas or liquid chromatography (GC-MS or LC-MS) andhistological methods (e.g. immunofluorescence (IF) andimmunohistochemistry (IHC) for GABA).

The person skilled in the art would be well aware of standard methodsfor measuring enzymatic activity, such as those described in EnzymeAssays: A Practical Approach (The Practical Approach Series) byEisenthal R and Danson M (2^(nd) edition, Oxford University Press, 2002)which is incorporated by reference herein.

So that the invention may be readily understood and put into practicaleffect, the following non-limiting Example is provided.

EXAMPLES Example 1—Effects of Triheptanoin on ALS in an Animal ModelMaterials and Methods

Animals

All experiments were approved by the University of Queensland's AnimalEthics Committee and followed the guidelines of the Queensland AnimalCare and Protection Act 2001. All efforts were made to minimize thesuffering and the number of animals used. Wild-type and hSOD1^(G93A)mice (B6.Cg-Tg(SOD1*G93A)1Gur/J, stock no. 004435, Jackson laboratory,Maine, USA), were generated by mating hSOD1^(G93A) males with C57B/L6wild-type females (University of Queensland). Mice were housed in a 12hour light, 12 hour dark cycle, and had free access to food and water.Experimenters were blinded to animal genotype (until the mice startedexpressing the ALS phenotype) and dietary interventions.

Dietary Intervention

Immediately after weaning, mice were placed on either a standard diet(SF11-027, Specialty Feeds, Wash., AUS) or a matched diet (SF11-028,Specialty Feeds) in which 35% of the calories were from triheptanoin oil(Sasol, Germany). All diets were matched in protein, mineral,antioxidant and vitamin content relative to their caloric densities(Thomas et al., 2012). Triheptanoin replaced sucrose, some of thecomplex carbohydrates and long chain fats in the diet.

Enzyme Assays

The cytosolic and mitochondrial fractions were isolated from thegastrocnemius muscle via homogenisation (Potter-Elvehjem tissue grinder)in cold isolation buffer (10 mM EDTA, 215 mM D-mannitol, 75 mM sucrose,0.1% BSA and 20 mM HEPES, pH 7.4). Samples were centrifuged at 700 g for10 minutes at 4° C. The supernatant was collected and centrifuged at10,500 g for 10 minutes at 4° C., the supernatant collected, and thepellet resuspended in 1 mL isolation buffer. The centrifugation processwas repeated, and the collective supernatant (cytosolic fraction) andresuspended pellet (mitochondrial fraction) stored at −80° C.

The activities of all enzymes were measured by continuousspectrophotometric assays at 25° C. on Sunrise Tecan microplate reader(Tecan, Mannedorf Switzerland). The reduction of NAD and NADP (OGDH andPGI activity, respectively) or the oxidation of NADH (PFK activity) wasmeasured by the change in absorbance over time at a wavelength of 340nm. All activity rates were corrected to milligrams of protein insamples, using the BCA protein Assay kit (Thermo Scientific, Illinois,USA).

OGDH Assay

The assay was adapted from Lai & Cooper, 1986. The assay mixturecontained 50 mM Tris-HCl (pH 7.4), 0.2 mM sodium CoA, 2 mM Nicotinamideadenine dinucleotide, 0.5 mM, thiamine pyrophosphate, 0.5 mM magnesiumchloride, 10 mM dithiothreitol and 10 μL of isolated mitochondria fromsample. Reactions were initiated by the addition of 10 mM oxoglutarateto measure the reduction of NAD.

PGI Assay

The activity of phosphoglucose isomerase (PGI) was measured by couplingthe reaction to NADP reduction via glucose 6-phosphate dehydrogenase.The assay mixture contained 100 mM Tris-HCl (pH 7.4), 0.6 mMnicotinamide adenine dinucleotide phosphate, 17.5 mM magnesium chloride,5 U/mL glucose 6-phosphate dehydrogenase (G8404, Sigma Aldrich), and 5μL of the cytosolic fraction from samples. The reaction was initiated bythe addition of 10 mM fructose 6-phosphate.

PFK Assay

The activity of phosphofructokinase was measured by coupling theproduction of fructose 1,6-bisphosphate from the reaction to the enzymesaldolase, α-glycerophosphate dehydrogenase and triosephosphate isomeraseto measure the oxidation of NADH. The assay mixture contained 80 mMTris-HCl (pH 7.4), 2 mM dithiothreitol, 3.6 mM adenosine triphosphate,0.6 mM nicotinamide adenine dinucleotide reduced, 20 mM magnesiumchloride, 6 U/mL aldolase (A8811, Sigma Aldrich), 1 U/mLα-glycerophosphate dehydrogenase and 5 U/mL triosephosphate isomerase(G1881, Sigma Aldrich) and 5 μL of the cytosolic fraction from samples.The reaction was initiated by the addition of 15 mM fructose6-phosphate.

Behavioral Testing and Observation

Animals underwent behavioral testing approximately 3-4 hours into thelight cycle. All behavioral testing was conducted in an environment withminimal stimuli so as to minimize any possible effects caused by changesin external stimuli. Animals were weighed before every test session.Mice were observed, and disease progression tracked and graded accordingto a neurological score sheet to ensure that any non-ALS related deathswere excluded from the study. The neurological score was adapted fromcriteria developed at the ALS therapy development institute (Gill etal., 2009). In accordance with ethical requirements, hSOD1^(G93A) micethat became too weak to reach the food hoppers were provided with wetchow on the floor of the cages. The endpoint of the study was defined asthe mouse being unable to right itself within 15 seconds after beingplaced on its back. Upon reaching end-stage or 25 weeks of age,transgenic mice and their respective wild-type littermates wereeuthanized with an overdose of pentobarbital (120 mg/kg, i.p., Provet,Australia). Tissues, including the m. gastrocnemius and tail, werecollected for subsequent analysis. To measure the time point when bodyweight loss started, we defined the day where a loss of more than 10% ofthe combined mean body weight from week 12 to 17 was observed and allsubsequent three measurements were ≤90% of the original mean weight.

Hind Limb Grip Strength Test

Hind limb grip strength tests were conducted twice a week using a T-barforce transducer (Ugo Basile, Italy). The animal was held by the tail,ensuring its hind limbs were gripping the T-bar before being pulleddownwards at a 60° angle. The reading on the force transducer was takenonly if both hind limbs released the bar at the same time. The averageof 10 trials per mouse was recorded each training session. To comparetime points of grip strength loss, the time point where a loss of morethan 30% of the combined mean grip strength of week 9 to 13 wasrecorded, if the subsequent 3 measurements were ≤70% of the originalaverage strength.

Rotarod Test

The rotarod tests were conducted with 10 repeats once a week using arotarod designed for mice (Ugo Basile, Italy). Animals were placed onthe rod and the rod was then rotated for 3 min at 25 revolutions permin. The time at which the animal fell off was recorded. We defined theage of balance loss on the rotarod when this time was zero.

Quantitative Genotyping

All mice were genotyped post-mortem by real time quantitative PCRaccording to a previously described procedure to assess relative copynumber of the hSOD1^(G93A) transgene (Alexander et al., 2004). Primersused were documented by the Jackson laboratory for genotyping withconventional PCR (http://jaxmice.jax.org). The final concentration offorward and reverse primers in each reaction was 0.4 μM for hSOD and 0.5μM for mouse interleukin 2 (mIl2) mixed with 5 ng genomic DNA and 5 μlof SYBR Green Mastermix (Applied Biosystems, CA, USA) The thermalprofile for the assay was an initial hot start of 95° C. for 10 minutes,followed by 40 cycles of 95° C. for 30 seconds, 60° C. for 1 minute and72° C. for 30 seconds (ABI 7900HT Fast Real-Time PCR system, AppliedBiosystems). Lastly the melt curve was generated by heating to 95° C.for 2 minutes, cooling to 60° C. for 15 seconds and a final 2% heatingramp to 95° C. for 15 seconds. Water was added to replace the DNA in thenegative controls. CT values were calculated by the Sequence DetectionSoftware 2.4 from the raw data. Copy number was then calculated usingthe equation documented below.

Copy number=(2^((CT) ^(hSOD1) ^(Log) ² ^(1.82))−^((CT) ^(mIL2) ^(Log) ²^(2.01)))2

RNA extraction, cDNA synthesis, Quantitative real time PCR assay.

After euthanasia, the gastrocnemius muscle was quickly removed andfrozen in liquid nitrogen. To extract RNA, muscle samples werepulverized in liquid nitrogen with a cold mortar and pestle, dissolvedwith TRI reagent (Life Technologies, CA, USA) and extracted according tothe manufacturer's instructions. Contaminating DNA was removed by DNaseI treatment and cDNA was synthesized using the Tetro cDNA synthesis kit(Bioline, London, UK) according to the manufacturer's instructions.

Expression of several metabolic genes was assayed (Gapdh, Pdha1, Ogdh,Sdha, Pcx, Gpt1, Gpt2, Pcca, Pccb and Mut) by quantitative real timePCR. All primers pairs (Table 1) were evaluated for efficiency using a 4fold serial dilution series of muscle cDNA. The derived slope of eachprimer pair was used to calculate the efficiency by applying theformula, 4^([(−1/slope)−1]*100). Reactions consisted with diluted cDNA,5 μl of SYBR Green Mastermix and 8 μM of forward and reverse primerseach, and were amplified after an initial hot start. Conditions forcycling and the melt curve were identical to those described forquantitative genotyping. Samples without reverse transcriptase treatmentwere assayed to ensure that samples were free from DNA contamination.The fold expression (ΔCT) of the gene of interest (goi) relative to thegeometric mean of housekeeping genes (Tbp, B2m and Hmbs) were calculatedwith a formula adapted from (Vandesompele et al., 2002) taking intoconsideration the individual efficiencies (E) of each primer pair.

${\Delta \; {CT}_{goi}} = {2^{-}\left\lbrack {\left( {{CT}_{goi}{Log}_{2}E_{goi}} \right) - {3\sqrt{\left( {{CT}_{TBP}{Log}_{2}2.03} \right)\left( {{CT}_{B\; 2\; m}{Log}_{2}2.03} \right)\left( {{CT}_{HMBS}{Log}_{2}1.86} \right)}}} \right\rbrack}$

Motor Neuron Counting

Mice were fed with control or triheptanoin diet from 35 days until 70days of age, when mice were anesthetized with pentobarbital (100 mg/kgi.p.), perfused with 0.9% saline followed by 4% paraformaldehyde. Spinalcords were removed and further fixed in 4% paraformaldehyde andsubsequently embedded in OCT compound and serial sections of 16 μm werecut from lumbar vertebrae. Sections were washed in cold PBS and stainedwith 0.1% thionin in acetate buffer (pH3.4). The motor neurons werecounted using stereological method as previously described (Banks etal., 2001; 2003; Forgarty et al., 2013). Briefly, motor neurons in thelateral motor column (LMC) from one side of the spinal cord were countedand only neurons that are large in size with darkly stained cytoplasm,pale nucleus and darkly stained nucleoli were counted (Clarke andOppenheim, 1995). Every 10^(th) section from lumbar 2 to lumbar 5regions was counted to get the total motor neuron count which was thendivided by the number of sections counted and multiplied by the totalnumber of sections containing the LMC.

Data Analysis

Statistics were performed with Graphpad Prism (version 5.03) using oneor two-way ANOVA followed by Bonferroni multiple comparisons post-hoctests for analysis of several groups. For the comparison of the onset ofbody weight loss and the area under the curve (AUC) for hind limb gripstrength, two-tailed unpaired t-tests were employed. Linear regressionanalysis with comparison of slope and intercept was used to compare ageof hind limb grip strength loss and body weight loss vs. transgene copynumber. Data are represented as mean±SEM.

Power analysis using the average standard deviations for onsets of lossof grip strength and balance on rotarod showed that n=5 was sufficientto observe changes between means by 2.3 and 1.5 weeks, respectively with80% power at the 0.05 significance level.

Results

Metabolic alterations in muscle of hSOD1^(G93A) mice. First we set outto confirm that energy metabolism is affected in hSOD1^(G93A) mice atthe mid and end disease stage. Specifically, we found that in the m.gatrognemicus, lactate levels are reduced at mid stage (110-130 days) by29%, indicating reduced glycolysis (FIG. 2, panel A). This was may beexplained by the observed reduction in the maximal activity ofphosphoglucose isomerase by 28.5% and phosphofructokinase by 53%decrease, while the oxidative stress-sensitive TCA cycleenzyme-oxoglutarate dehydrogenase maximal activity was reduced by 25% (*p<0.05 t-tests. N=5-8 mice/group, FIG. 2, panel B). In contrast at endstage, muscle glucose levels were elevated 1.9-fold, suggesting loss ofglycolysis.

Triheptanoin delays the onset of loss of hind limb grip strength andbalance in hSOD1^(G93A) mice. One of the characteristics of ALS inhSOD1^(G93A) mice is the progressive loss of muscle mass and strength.Thus, hind limb grip strength tests were used to assess the course ofdisease progression in mice on either a control or triheptanoin diet.There was no observable difference between the mean hind limb gripstrength of the wild-type mice on the control diet (n=12) when comparedto those on the triheptanoin diet (n=15; all hSOD1^(G93A) mice includinglower transgene copy number). Mean hind limb grip strengths for controlfed and triheptanoin fed wild-type mice consistently fell between 300and 600 mN (FIG. 3A).

During the course of the experiments, it was observed that somehSOD1^(G93A) mice developed a loss of hind limb grip strength after 22weeks of age. Analysis of hSOD1^(G93A) transgene copy number revealedthat a subpopulation of hSOD1^(G93A) mice had only 12-17 copies of theSOD1 transgene. Assessing all mice (FIG. 3E), the linear regressionsbetween the age of strength loss beginning against transgene copynumbers are significantly different between the groups fed control diet(R²=0.89) vs. triheptanoin (R²=0.91). Specifically, the x and yintercepts are different between the two regression lines (p<0.001)(indicating that triheptanoin effectively delays onset of grip strengthloss), but not the slopes (p=0.90).

For the following analyses, mice with only 12-17 copies of the SOD1transgene were excluded from the analysis of grip strength and geneexpression. Mice with 19-24 copies of the transgene were combined as onegroup with high copy transgene numbers. The grip strength ofhSOD1^(G93A) mice on both diets never exceeded 400 mN (FIG. 3B). Thetime course of reduced hind limb grip strength was significantlydifferent between high copy number hSOD1^(G93A) mice on the control diet(n=5) when compared to those on the triheptanoin diet (FIG. 3B, n=8;p=0.04, two way ANOVA). Bonferroni's multiple comparisons testsindicated that at 18 and 19.5 weeks of age, triheptanoin fedhSOD1^(G93A) mice had higher hind limb grip strength when compared tocontrol diet fed hSOD1^(G93A) mice (FIG. 3B, p<0.05). The area under thecurve for the grip strength over time for each mouse on triheptanoin wasincreased by 38% relative to control diet (p=0.024, t-test, FIG. 3C).hSOD1^(G93A) mice on the control diet began to lose hind limb gripstrength at 16.5 weeks of age. The time of onset of the loss of hindlimb grip strength was delayed by 2.8 weeks in hSOD1^(G93A) mice fedtriheptanoin (FIG. 3D). In the rotarod test, behavior of thehSOD1^(G93A) mice varied widely, many mice seemed not to be “motivated”to learn how to balance on the rod and no satisfying presymptomaticbaseline was reached. Therefore we could only assess the time point whenmice were unable to stay on the rod. Triheptanoin delayed the time ofonset of loss of balance by 1.6 weeks (p=0.0016; FIG. 3F).

Body weight loss and survival in hSOD1^(G93A) mice with and withouttriheptanoin. Body weight is another indicator of disease progression inhSOD1^(G93A) transgenic mice. When compared to wild-type mice on thecontrol diet (n=12), wild-type mice on the triheptanoin diet (n=15)gained weight at a slower rate and were lighter when culled (p<0.0001,FIG. 3G). At 14 weeks of age, triheptanoin fed wild-type mice wereapproximately 3 g lighter than control fed wild-type mice (p<0.05). Thisweight difference increased to approximately 4 g at 22 weeks of age.

Mice carrying 19-24 copies of the hSOD1 transgene on the control diet(n=5) and the triheptanoin diet (n=8) were lighter when compared towild-type mice on the control diet (p<0.0001). There were nostatistically significant differences between the hSOD1^(G93A)transgenic mice on different diets over the full time period. However,compared to control diet, triheptanoin fed hSOD1^(G93A) mice showed atrend of reduced body weight gain from 7-16 weeks. During the diseaseprocess after 20 weeks of age, the body weight of hSOD1^(G93A) mice onthe control and triheptanoin diet became similar (FIG. 3G). The onset ofbody weight loss in hSOD1^(G93A) mice was delayed by triheptanoinfeeding by 1.6 weeks (FIG. 3F, p=0.007, unpaired two-tailed t-test).

Only a small number of hSOD1^(G93A) mice reached end-stage of disease,therefore our survival analysis is of limited power. No differences wereseen in the number of days taken to reach end-stage when comparingtriheptanoin (n=7) to control fed (n=5) hSOD1^(G93A) mice (174.9±3.5 vs.172.4±3.9, p=0.653), indicating that in this small study, triheptanointreatment did not alter survival.

Motor neuron counts. We assessed the number of motor neurons bystereological counting between L2 and L5 in mice at 70 days, whichcorresponds to onset of disease. The statistically significant loss of37% of motor neurons in hSOD1^(G93A) vs. wild type mice fed control diet(CON, n=3-4) was alleviated by feeding triheptanoin, as neuron numberswere not significantly different from wild type mice (n=4, FIG. 4).

Gene expression studies. To evaluate the extent to which TCA cyclemetabolism or anaplerosis may be impaired in the gastrocnemius muscle ofmid copy number hSOD1^(G93A) mice at 10 weeks at symptom onset,quantitative real time PCR was used to compare the expression of genesinvolved in these pathways. The hSOD1^(G93A) mice in the two diet groupshad similar hSOD1^(G93A) copy numbers, with 12-17 (average 14.4 copies)in the Con diet and 12-20 (average 15.75) copies in the triheptanoin-fedgroup (p=0.51 unpaired t-test). We chose to study the expression ofenzymes involved in glycolysis (glyceraldehyde-3-phosphatedehydrogenase—Gapdh), the TCA cycle (2-oxoglutarate and succinatedehydrogenases, FIG. 5) and anaplerotic pathways of the muscle. Thelatter include pyruvate carboxylase (Pcx) producing oxaloacetate,glutamic pyruvic transaminase 1 and 2 (Gpt1 and 2, FIG. 5), and theenzymes of the propionyl-CoA carboxylation pathway (FIG. 6), namely thealpha and beta subunit of propionyl-carboxylase (Pcca and Pccb) andmethylmalonyl mutase (Mut), which together metabolize propionyl-CoA tosuccinyl-CoA. Triheptanoin fed mice were included in this analysis toinvestigate if triheptanoin could alleviate any alterations observed.

In 10 week old pre-symptomatic hSOD1^(G93A) mice, quantitative real timePCR showed that when compared to wild-type mice, the expression ofseveral dehydrogenases and methyl-malonyl mutase was reduced. Namely, wefound statistically significant reductions for the mRNA levels of the A1subunit of pyruvate dehydrogenase (Pdha1) by 24%, 2-oxoglutaratedehydrogenase (Ogdh) by 30%, the subunit A of succinate dehydrogenase(Sdha) by 23% and methyl-malonyl mutase (Mut) by 27.5% in hSOD1^(G93A)mice (FIGS. 3, 5; all p<0.05 in post-test). Triheptanoin feedingprotected hSOD1^(G93A) mice from changes in the expression of pyruvateand succinate dehydrogenases, but not 2-oxo-glutarate dehydrogenase andmethylmalonyl mutase. No alterations of mRNA levels were found in theother investigated genes, including glycolytic Gapdh and the genesinvolved in anaplerosis, Gpt1 and 2, Pcca and Pccb (FIGS. 5 and 6).Again triheptanoin feeding prevented some of the mRNA level reductions,indicating that it is effectively preserving the muscle and itsmetabolism.

Discussion Effects of Triheptanoin and Clinical Importance

One of the findings of this study is that hSOD1^(G93A) mice showmetabolic alterations (summarized in FIG. 7) in glycolysis and TCA cycleenzyme activity. These metabolic deficiencies can be mechanisticallyaddressed by feeding triheptanoin, because it provides an alternativefuel to glucose and will improve TCA cycling and thereby oxidation ofany fuel, including glucose.

Indeed, triheptanoin attenuated reductions in the gene expression ofenzymes involved in TCA cycling and delayed symptoms of diseaseoccurrence and disease progression in hSOD1^(G93A) mice. This wasobserved as a delay in motor neuron death, in the onset of the loss ingrip strength and loss of balance on the rotarod, a delay in the loss ofbody weight, and the reversal of the reduced expression of metabolismgenes. Our data show a clear improvement in the condition of thehSOD1^(G93A) mice in which triheptanoin feeding was initiated prior tothe onset of disease symptoms. On the other hand, presymptomatichSOD1^(G93A) mice already show loss of crural flexor neurons (Ngo etal., 2012).

As a medium chain triglyceride, triheptanoin quickly provides heptanoicacid to the blood, which can enter all tissues and mitochondria bydiffusion without involvement of carrier systems. Also, C5 ketones arequickly produced by the liver and they can enter most tissues viamonocarboxylate transporters. Therefore, it is expected that anaplerosisvia this pathway will begin quickly after treatment initiation.

Our power analyses regarding loss of grip strength and balance show thatour study used adequate numbers of mice regarding these analyses ofmotor symptoms. From this our data show clear preservation of musclefunction (disease modification) despite potential confounding effects onbody weight. These data are very promising and warrant phase I clinicalassessment of safety in ALS patients.

Metabolic Changes in hSOD1^(G93A) Mouse Muscle

The decrease in lactate levels in muscle was interpreted as a decreasein glycolysis, because the level of lactate correlates strongly withthose of pyruvate. We assumed that pyruvate levels are declined due tolowered activity of glycolytic enzymes PGI and PFK and in additionlowered activity of OGDH, which indicates slowing of the TCA cycle.Furthermore, our quantitative real time PCR data show that when comparedto healthy wild-type mice, the expression of several enzymes involved inglycolysis, TCA cycle and anaplerosis were significantly reduced in thegastrocnemius muscle of hSOD1^(G93A) mice at 10 weeks of age (FIGS. 5,6) a time when hind limb grip strength is still normal (FIG. 3B). Takentogether the specific downregulation of these enzymes suggest that TCAcycling is slowed in the muscle early before symptom onset and thatinsufficient ATP is produced for survival of tissue. Moreover, we foundreduced mRNA levels of the main anaplerotic enzymes of muscle, the twoglutamic pyruvic transaminases (gpt1 and 2), at 25 weeks, suggestingdiminished levels of TCA cycle intermediates (data not shown).

In addition to slowing the disease process, triheptanoin prevented thedownregulation of several enzymes which were reduced in thegastrocneminus muscles from hSOD1^(G93A) when compared to wild-typemice. This finding implies that normalization of energy metabolism bytriheptanoin may prevent downregulation of certain metabolism genes,which in turn will help to maintain a healthy metabolism to optimizeenergy supply and survival of tissue.

CONCLUSION

This study reveals that triheptanoin is a promising new treatmentapproach for ALS. Our data support initial clinical safety andtolerability trials of triheptanoin in ALS patients.

Throughout the specification, the aim has been to describe someembodiments of the invention without limiting the invention to any oneembodiment or specific collection of features. Various changes andmodifications may be made to the embodiments described and illustratedwithout departing from the present invention.

The disclosure of each patent and scientific document, computer programand algorithm referred to in this specification is incorporated byreference in its entirety. To the extent that any definitions indocuments incorporated by reference are inconsistent with thedefinitions provided herein, the definitions provided herein arecontrolling.

REFERENCES

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1. A method of treating an animal with a neurodegenerative and/orneuromuscular disease, disorder or condition, wherein said methodincludes the step of administering a therapeutically effective amount ofone or more anaplerotic agents to said animal, to thereby treat theneurodegenerative and/or neuromuscular disease, disorder or condition insaid animal.
 2. (canceled)
 3. The method of claim 1, wherein theneurodegenerative and/or neuromuscular disease, disorder or condition isa motor neuron disease (MND).
 4. The method of claim 3, wherein the MNDis amyotrophic lateral sclerosis (ALS), primary lateral sclerosis (PLS),progressive muscular atrophy (PMA), progressive bulbar palsy (PBP),pseudobulbar palsy, or spinal muscular atrophy (SMA).
 5. The method ofclaim 3, wherein the MND is ALS.
 6. The method of claim 1, wherein theone or more anaplerotic agents are selected from the group consisting ofglutamate, glutamine, pyruvate and one or more precursors ofpropionyl-CoA.
 7. The method of claim 6, wherein the one or moreprecursors of propionyl-CoA are selected from the group consisting of anuneven chain fatty acid, a triglyceride, a C5 ketone body, aphospholipid, a branched chain amino acid and combinations thereof. 8.The method of claim 7, wherein the triglyceride is one or more compoundsof Formula I:

wherein R₁, R₂ and R₃ are independently selected from alkyl, alkenyl oralkynyl.
 9. The method of claim 8, wherein R₁, R₂ and R₃ areindependently selected from C₁ to C₂₀ alkyl, alkenyl or alkynyl.
 10. Themethod of claim 9, wherein R₁, R₂ and R₃ are the same and are selectedfrom the group consisting of C₅, C₇, and C₉ alkyl.
 11. The method ofclaim 9, wherein the compound of Formula I is selected fromtripentanoin, triheptanoin and trinonanoin.
 12. The method of claim 11,wherein the compound is triheptanoin.
 13. The method of claim 1, whereinthe one or more anaplerotic agents are provided to the animal in anamount comprising at least about 5% of the dietary caloric intake forthe animal.
 14. The method of claim 13, wherein the one or moreanaplerotic agents are provided to the animal in an amount comprising atleast about 20% of the dietary caloric intake for the animal.
 15. Themethod of claim 14, wherein the one or more anaplerotic agents areprovided to the animal in an amount comprising at least about 30% of thedietary caloric intake for the animal.
 16. The method of claim 15,wherein the one or more anaplerotic agents are provided to the animal inan amount comprising at least about 35% of the dietary caloric intakefor the animal.
 17. The method of claim 1, wherein the animal is anadult animal.
 18. The method of claim 1, wherein the animal is a mammal.19. The method of claim 18, wherein the mammal is a human.
 20. Themethod of claim 1, wherein the therapeutically effective amount of oneor more anaplerotic agents is administered orally. 21-37. (canceled)